A cluster of mesopontine GABAergic neurons suppresses REM sleep and curbs cataplexy

Physiological rapid eye movement (REM) sleep termination is vital for initiating non-REM (NREM) sleep or arousal, whereas the suppression of excessive REM sleep is promising in treating narcolepsy. However, the neuronal mechanisms controlling REM sleep termination and keeping sleep continuation remain largely unknown. Here, we reveal a key brainstem region of GABAergic neurons in the control of both physiological REM sleep and cataplexy. Using fiber photometry and optic tetrode recording, we characterized the dorsal part of the deep mesencephalic nucleus (dDpMe) GABAergic neurons as REM relatively inactive and two different firing patterns under spontaneous sleep–wake cycles. Next, we investigated the roles of dDpMe GABAergic neuronal circuits in brain state regulation using optogenetics, RNA interference technology, and celltype-specific lesion. Physiologically, dDpMe GABAergic neurons causally suppressed REM sleep and promoted NREM sleep through the sublaterodorsal nucleus and lateral hypothalamus. In-depth studies of neural circuits revealed that sublaterodorsal nucleus glutamatergic neurons were essential for REM sleep termination by dDpMe GABAergic neurons. In addition, dDpMe GABAergic neurons efficiently suppressed cataplexy in a rodent model. Our results demonstrated that dDpMe GABAergic neurons controlled REM sleep termination along with REM/NREM transitions and represented a novel potential target to treat narcolepsy.


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Surgeries of implanting fibers and electrodes 12 Three weeks after injection, the mice used for in vivo studies were chronically implanted with EEG/EMG 13 electrodes for polysomnographic recordings under isoflurane anesthesia (5% induction, 1.5% maintenance).
14 The electrodes consisted of two stainless steel screws connected to EEG Teflon-coated wires, which were 15 inserted through the skull, and two EMG Teflon-coated wires, which were bilaterally placed into both trapezius 16 muscles. All of the electrodes were fixed to the skull with dental cement and attached to a microconnector 1 .

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For fiber photometry, the tips of fiber-optic cannulas (200-μm diameter; Newton Inc., Hangzhou, China) were 18 inserted into the viral-injection sites before implantation of the electrodes. For optogenetic experiments, the 19 tips of fiber-optic cannulas were inserted at 0.2 mm above the viral-injection sites or axonal-terminal locations 20 before implantation of the EEG/EMG electrodes. The scalp wound was closed with surgical sutures, and each 21 mouse was kept in a warm environment until it resumed normal activity as previously described 1 . After 22 surgery, the mice were housed separately for approximately 7 days for recovery.

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The mice were first connected to the sleep recording equipment to habituate for 72 h before the formal video-25 polysomnography recordings. A cable with a slip ring (Kissei Comtec Co., Ltd, Japan) was connected to mice 26 in the cage for 7 days before the initiation of EEG and EMG recordings. The EEG/EMG signals were 27 amplified, filtered, and digitized as previously described 2 .

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RV-based retrograde tracing and cell counting 29 An RG-deleted RV strategy for retrograde tracing has been reported to mark monosynaptic inputs to 30 specifically selected starter cells. This method has been successfully used in previous studies by our group.

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For cell mapping of neurons, neuronal somata were quantified semi-automatically via ImageJ software as 32 previously described 3 . We divided each brain into six general structures, which together encompassed more 33 than 50 specific brain regions containing all DsRed-labeled neurons throughout the brain (n = 4 mice).

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Data analysis of Fiber photometry 35 Photometry data were exported to Matlab Mat files from Spike2, and then analyzed by customized Matlab 36 software (Matlab, 2016a, MathWorks, United States) as described in our previous study. For each session, 37 3 the photometry signal F was converted to ΔF/F by calculating ΔF/F = (F−Fmean)/Fmean, where Fmean is the 1 average fluorescence signal in each recording episode. For the analysis of mean fluorescence signal, we 2 derived the value of the photometry signal by calculating the Z-score = (F−Fmean)/σ, where σ is the standard 3 deviation of the fluorescence signal. For the sleep-wake analysis, we recorded data for 4-6 h per mouse each 4 day and calculated the averaged Z-score during all vigilance states. To analyze the state transition, we 5 determined each state transition and aligned the Z-score in a ± 30 s window before and after the switch point.

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Multichannel recording by optic tetrodes 7 The tetrode wire tips were plated with gold (Cyanida Gold solution, SIFCO Selective Plating) to adjust the 8 impedance to 500-800 kΩ. The wire tips were 0.5 mm longer than the optical fiber end to achieve efficient 9 photostimulation of the recorded neurons in vivo. For implantation, the electrode was slowly advanced into the

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To identify ChR2-tagged neurons, laser pulse trains (20 Hz or 40 Hz, with a duration of 1 and 0.5 s, 17 respectively) were delivered every 1 or 2 min. A unit was identified as GABAergic if spikes were evoked by 18 laser pulses at short first-spike latency (<8 ms) and the waveforms of the laser-evoked and spontaneous 19 spikes were highly similar. Spikes during the laser pulse trains were excluded to compute the mean firing rate 20 of each neuron in each brain state.

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To test whether the probability of sleep has changed after manipulation of these neurons' activity, we quantify 23 the probability of stages transition from an identified initial state to the following dominated states. The  1 were cut on a vibratome (VT-1200, Leica Microsystems), and only those including targeted brain nuclei were 2 selected and incubated in ACSF (32°C) for 30 min. The composition of the ACSF was as follows (in mM): 25 3 glucose, 119 NaCl, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 1 MgCl2, and 2 CaCl2 (at 32°C). All solutions were 4 made fresh and were saturated with 95% O2 and 5% CO2 before use.

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For whole-cell recordings, slices were placed in the recording chamber after 30-min recovery, and were 6 perfused with oxygenated ACSF (30-32°C, 1-2 mL/min). Only healthy neurons were recorded, as confirmed 7 by infrared differential-interference-contrast and fluorescence microscopy. In the current-clamp mode, we 8 acquired the electrophysiological properties of the recorded neurons, including the resting membrane potential 9 and action potentials. In the voltage-clamp mode, we held the potential at −70 mV for whole-cell recordings.

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Immunohistochemistry and immunofluorescence 18 Adult mice were deeply anesthetized and transcardially perfused with cold normal saline followed by 4% 19 paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). The brain was post-fixed for 2 h and then 20 cryoprotected in 30% sucrose. After embedding in optimal-cutting-temperature compound, the brain was   In situ hybridization, with either glutamate decarboxylase 1 (GAD1, 67 kDa) or Vglut2 mRNA, was performed 34 via digoxigenin riboprobes in brain sections. The brain sections were placed onto slides and were surrounded 35 by water-repellent traces. Subsequently, the slides were post-fixed in 4% PFA for 20 min. In situ hybridization 36 was processed as previously described 4,5 . DNA templates for the in situ hybridization probes were obtained 37 by PCR from either wild-type-embryo or P0-mouse cDNA libraries. All of the buffers contained 0.1% RNase 38 5 inhibitor (diethyl pyrocarbonate, DEPC, B600154, Sangon Biotech). Finally, the sections were mounted on 1 slides, dried, and coverslipped with Vectamount (Vector Laboratories). Images were acquired using a Leica 2 confocal system or an Olympus IX71 microscope.

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For DsRed immunohistochemistry combined with GAD1 or Vglut2 mRNA in situ hybridization, brain 4 sections were placed onto slides and in situ hybridization was performed following completion of DsRed 5 immunohistochemistry. All of the buffers contained 0.1% of RNase inhibitor.

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Fluorescence in situ hybridization (FISH) was performed using RNAscope Multiplex Fluorescent v2 7 Assays according to the manufacturer's instructions (Advanced Cell Diagnostics). Cells with more than 10 8 fluorescent dots in the cytoplasm were considered to be positively labeled.                    14 15